Prevention of and therapy for radiation toxicity of normal tissues using drugs which block il-1 activity

ABSTRACT

A method for the prevention of and therapy for radiation pneumonitis, dermatitis, soft tissue fibrosis and central nervous system toxicity in patients undergoing therapeutic radiation. In addition, the invention provides for pre-treatment of those responding to nuclear bio terrorism or other nuclear or radiological accidents. Thus, with the present invention, subjects may be treated in order to prevent toxicity from nuclear bio terrorism or other nuclear or radiological accidents. More particularly, we have discovered a method for prophylactically treating radiation toxicity in normal tissue of subject comprising administering an anti-radiation toxicity effective amount of a cytokine blocking agent through the subject. More specifically, we have discovered a method for prophylactically treating radiation pneumonitis, dermatitis, soft tissue fibrosis or central nervous system toxicity in a subject comprising administering an anti-radiation pneumonitis, dermatitis, soft tissue fibrosis or central nervous system toxicity effective amount of a cytokine blocking agent to the subject.

BACKGROUND

There are few if any treatments for radiation exposure that havequantitative dose modifying benefits when given hours a day afterexposure. Similarly, even those drugs that have benefits when givenbefore radiation, typically are ethicacious only for a few hours andhave transient side effects that prevent the subject from full function,for example, hypotension and peripheral neuropathy.

Radiation-induced soft tissue fibrosis is a consequence of acute andchronic inflammatory responses. While modern radiation techniques haveimproved therapeutic gain and reduced the incidence of severeradiation-induced fibrosis, radiation-related side effects still occurwhen aiming for optimal tumor control. It has been shown thatradiation-induced soft tissue damage is expected in about 10% ofpatients when radiation is optimized to achieve 90% tumor control.

Soft tissue fibrosis occurs in the late stage of radiation-inducedtissue damage. It is caused by multiple factors and is poorlyunderstood. However, the early stage of radiation-induced soft tissuedamage is characterized by infiltration of various inflammatory cellsand overproduction of cytokines. The late stage is pathologicallycharacterized by active fibroblast proliferation with atypicalfibroblasts, and excessive extracellular matrix production. Radiationinjury is similar in some ways to normal tissue injury. Surgical injury,for example, is a process that features a relatively short period ofbrisk cytokine production, angiogenesis, fibroblast, and epithelial cellproliferation. The atypical proliferation results in granulation, whichabruptly stops, allowing mature scar to develop. IL-1 is an importantsignal controlling this process. Radiation-induced soft tissue fibrosishas many of the same features of normal tissue repair, but is less briskand may remain active for years at subclinical levels. The continuousinflammation results in continuously active deposition of collagen.

Radiation pneumonitis is a distinct clinical entity that differs fromother pulmonary symptoms such as allergic pneumonitis, chemicalpneumonitis, or pneumonia by various infectious agents. Recent researchhas supported the mechanism of cellular interaction between lungparenchymal cells and circulating immune cells mediated through avariety of cytokines including pro-inflammatory cytokines, chemokines,adhesion molecules, and pro-fibrotic cytokines. Identifying reliablebiomarkers for radiation pneumonitis will allow identifying individualsat risk for pneumonitis before or during the early stage of therapy.

Radiation pulmonary injury manifested as subacute pneumonitis and latefibrosis has long been recognized in patients receiving radiotherapy tothe chest region. Lung injury by radiation is a major obstacleprohibiting the high dose radiation required for eradicating cancer ofthe thoracic region. Radiation pneumonitis is a distinct clinical entityand there has been increasing awareness and recognition of its impact onthe treatment of thoracic malignancy. It manifests unique clinical andradiographic characteristics that separate it from other pulmonarysymptoms such as allergic pneumonitis, chemical pneumonitis, orpneumonia by various infectious agents of viral, bacterial, fungal, orparasitical origins.

Radiation pneumonitis is a type of inflammatory response of the lungtissue in response to radiation insult. Indeed, at the cellular level,radiation pneumonitis is characterized by lymphocytic alveolitis, aresult of inflammatory infiltrates of mononuclear cells from thevascular compartment into the alveolar spaces. As expected at sites ofinflammation, an active interaction between cellular and humoral factorsare involved including immune cells, parenchymal cells, macrophages,chemokines, adhesion molecules, lymphocytes, inflammatory cytokines andfibrotic cytokines. Research in radiation pulmonary injury has supportedinvolvement of inflammatory cytokines, chemokines, and fibroticcytokines. Although investigation of adhesion molecules in radiationlung injury is still underway, these molecules are expected to beinvolved to serve as prerequisites for leukocyte adhesion to endothelialcells of blood vessels and consequently for transmigration to tissues atsites of inflammation. At the time of clinical symptoms, radiographicinfiltrates are often observed in lung volumes, which generally conformto the radiation treatment ports on chest radiographs. The alveolarspaces are filled with patchy infiltrates on chest CT scans and thepatients often experience worsening dyspnea. These mononuclearinfiltrates may be cleared from alveolar spaces rapidly in response tosteroids, likely due to rapid apoptosis of lymphocytes by steroids, andpatients often experience marked improvement of dyspnea. With longerfollow-up, almost all patients develop radiographic evidences of lungfibrosis.

While current fast-developing new techniques have significantly improvedradiotherapeutic gains, radiation-related normal tissue damage stillremains unavoidable especially when aiming for optimal tumor control.Normal tissue tolerance, in particular, soft tissue fibrosis, is one ofthe major dose-limiting factors influencing radiation therapy. It hasbeen reported that radiation-induced soft tissue damage is expected inten percent of patients when radiation dose is optimized to maximumtumor control. Therefore, a better understanding of the molecular basisof radiation-induced normal damage could provide an effective means forthe prevention, or even reversal of radiation-related complications inthe clinical radiotherapy. Furthermore, due to the unsatisfactoryoutcomes of present combination of radiotherapy and chemotherapy,especially with multiple-areas and prolong schedule procedure, muchemphasis also are needed to placed on developing better and lessside-effects treatment procedure for normal tissue protection.

SUMMARY OF THE INVENTION

We have discovered that IL-1 is a major contributor to acute and lateradiation complications to the bone marrow, bowel, and lungs and softtissues. We have shown that humans that have high circulating levels ofIL-1 before any radiation is delivered develop radiation pneumonitis. Inaddition, we have found that the absence of IL-1 alpha results in a lowpropensity for the development of fibrosis following radiation. However,we have also discovered that the elevation of IL-1 persists or rises atlater times after radiation.

We have further found that blocking IL function with circulatingproteins or drugs is a useful method for the prevention of toxicity tonormal tissue and is ethicacious after radiation for the prevention ofthe progression of toxicity over time.

As a result, the present invention provides for the prevention of andtherapy for radiation pneumonitis, dermatitis, soft tissue fibrosis andcentral nervous system toxicity in patients undergoing therapeuticradiation. In addition, it provides for pre-treatment of thoseresponding to nuclear bio terrorism or other nuclear or radiologicalaccidents. Thus, with the present invention, subjects may be treated inorder to prevent toxicity from nuclear bio terrorism or other nuclear orradiological accidents. More particularly, we have discovered a methodfor profallactically treating radiation toxicity in normal tissue of asubject comprising administering an anti-radiation toxicity effectiveamount of a cytokine blocking agent through the subject.

More specifically, we have discovered a method for profallacticallytreating radiation pneumonitis, dermatitis, soft tissue fibrosis orcentral nervous system toxicity in a subject comprising administering ananti-radiation pneumonitis, dermatitis, soft tissue fibrosis or centralnervous system toxicity effective amount of a cytokine blocking agent tothe subject.

We have further discovered that the administration of an anti-radiationinduced soft tissue effective amount of a COX-2 enzyme inhibitorsignificantly reduces the amount of tissue damage due to radiation.

We have investigated the role of specific COX-2 inhibitors (Celebrex) inradiation induced soft tissue damage, and explored the relationshipbetween chemokine and its receptor mRNA expression and radiation-inducedskin damage in mammary tumor-bearing mice. Here we report that 50 mg/kgCelebrex, given daily with gavage for 15 doses in three weeks,significantly reduced single dose of radiation (60 Gy) induced normalskin damage in MCa-35 mammary tumor-bearing mice. Decreased skin damagesare associated with the reduction of the radiation-induced chemokines,Rantes, MCP1, and their related receptor mRNA expression in skin, butnot in tumor tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time scale of the currents of pneumonitisat various time points after radiation;

FIG. 2 is a serious of graphs showing the absolute cytokine level andrelative cytokine changes between groups with and without radiationpneumonitis.

FIG. 3 shows the results of circulatory cytokine changes of severalcytokines;

FIG. 4 shows plasma levels of Monocyte Chemotactic Protein 1;

FIG. 5 depicts typical changes in gross appearance after radiation ofskin;

FIG. 6 Shows a histological changes at various times after radiation;

FIG. 7 graphically depicts the basil levels of IL-B mRNA in mouse skin

FIG. 8 graphically depicts the basil levels of IL-B mRNA in mouse skin

FIG. 9 depicts the circulating IL-1β tissue mRNA expression;

FIG. 10 depicts IL-1α mRNA expression in muscle;

FIG. 11 depicts the effects of radiation on IL-1 Ra mRNA in muscle;

FIG. 12 depicts skin lesions in mice after 20 days of radiation

FIG. 13 depicts inflammation and cellular component infiltration in thedermis in Celebrex treated mice

FIG. 14 summarizes the effects of Celebrex on radiation-induced mRNAexpression of chemokines

FIG. 15 depicts the infiltration of inflammatory cells in the derma ofCelebrex-treated mice.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Materials and Methods: Prospective blood sampling, scoring ofrespiratory symptoms, and chest imaging were conducted for patientsreceiving thoracic radiation for malignancy. Serial plasma specimenswere analyzed for circulating cytokine changes before, during radiation,and up to 12 weeks post-radiation. Radiation pneumonitis was diagnosedusing NCI common Toxicity Criteria. Cytokine analysis was assayed forinterleukin a (IL-1α), interleukin 6 (IL-6), Monocyte ChemotacticProtein 1 (MCP-1), E-Selectin, L-Selectin, Transforming Growth Factor β1(TGF-β1), and Basic Fibroblast Growth Factor (bFGF) using Enzyme LinkedImmmunosorbant Assay (ELISA).

Methods

Patient Characteristics

Patients who were to receive thoracic radiation for malignancy wereeligible. Blood, thoracic imaging, and respiratory symptom scoring werecollected prospectively. Twenty-four patients had follow-up longer than12 months and their characteristics are shown in Table 1. TABLE 1Patient Characteristics Pneumonitis (NCI CTC Grade) Grade 1 (Radio-Grade 2 graphic (Sympto- All No Infiltrates matic Patients PneumonitisOnly) Pneumonitis) No. of Patients 24 5 (20.8%) 6 (25%) 13 (54%) Age: 63(40-80) Median (range) Sex (M:F) 11:13 2:3 3:3  6:7 Race (W:H) 23:01 5:06:0 12:1 Histology Squamous cell  5 (20.8%) 2 1  2 Andenocarcinoma 11(45.8%) 1 3  7 Andenosquamous  1 (4.2%) 0 0  1 Non-small nos  3 (12.5%)1 2  0 Small cell  3 (12.5%) 1 0  2 Thymoma  1 (4.2%) 0 0  1 Total 24 56 13 Tumor present Yes 21 (87.5%) 5 3 13 No  3 (12.5%) 0 3  0 Clinicalstage I  3 (12.5%) 0 2  1 II 0 III 15 (62.5%) 2 4  9 IV  3 (12.5%) 2 0 1 Limited small cell  3 (12.5%) 1 0  2 Chemotherapy No  6 (25%) 1 2  3Yes  3:15 (75%) 1:2 0:4  2:9 (ncocadjuvant: concurrent)Abbreviations: NCI CTC, National Cancer Institute common toxicitycriteria; nos, not otherwise specified.Clinical and Radiographic Evaluation

History and physical examinations emphasizing the respiratory symptomswere performed periodically. Clinical evaluation for pulmonary symptomswas evaluated and graded using the LENT/SOMA scoring system for thelung. This system includes the RTOG treatment side effect scoring ofsubjective clinical symptoms, and an objective assessment of serialchest X-rays and CT scan changes. Pneumonitis grading was also definedaccording to NCI Common Toxicity Criteria.

Circulating Cytokinie Analysis

Plasma samples were collected before therapy and weekly, during therapy.Specimens were collected in sodium heparin as well as EDTA up to 12weeks post-therapy. Platelet-free plasma was produced by centrifugationat 1200 rpm at 0° C. for 10 minutes. The plasma was stored in aliquotsat −20° C. Heparinized plasma was used for the analysis of mostcytokines and EDTA plasma was used for the analysis of bFGF. Cytokineswere analyzed using Enzyme-Linked Immunosorbent Assay (ELISA). Themethodology of ELISA analysis was according to manufacturers'instructions as previously described.

Twenty-four patients had clinical follow-up longer than 12 months afterradiation. Thirteen developed symptomatic pneumonitis (NCI grade 2). Thepeak incidence of symptoms was between 6- and 13 weeks postradiotherapy. Six patients had only radiographic infiltrates. (NCI grade1). Five patients did not have clinical or radiographic pneumonitis.Both IL-1α and IL-6 levels were significantly higher before, during, andafter radiation for those who developed pneumonitis. The pattern ofchanges of MCP-1, E-Selectin, L-Selectin, TGF-β1, and bFGF varied butnone of these cytokines correlated with radiation pneumonitis.

Analysis of a panel of circulatory cytokines with different putativefunction in radiation pulmonary injury showed that pre-treatment IΛ-1αand IL-6, as well as mid and post-treatment levels were significantlyhigher for patients who subsequently developed radiation pneumonitis.

Radiation Pneumonitis

Symptomatic radiation pneumonitis is characterized by an annoying coughthat is either non-productive or with clear sputum. This period isgenerally accompanied by markedly worsening dyspnea in an otherwisehealthy appearing individual. Generally there are also radiographicinfiltrates on chest x-ray and CT scan that usually conforms toradiation ports. The individual in general is afebrile or has alow-grade temperature, and is without an increase of blood neutrophilcounts. Clinical symptoms are rapidly relieved with low dose steroidtreatment. Of the 24 patients with follow-up longer than 12 months, 13developed clinical symptoms consistent with radiation pneumonitis (NCIgrade 2 pneumonitis). Six had radiographic infiltrates only, withoutclinical symptoms (NCI grade 1). Five did not have either radiographicinfiltrates or clinical symptoms. The timescale of occurrence ofpneumonitis is shown in FIG. 1. As demonstrated in FIG. 1, asymptomaticinfiltrates occurred at random time points after radiation, whilesymptomatic pneumonitis occurred most commonly between 6 weeks and 13weeks after completion of radiation. For all symptomatic pneumonitis,the first episodes all occurred within 6 months post-radiotherapy. Theoutliers beyond 6 months in FIG. 1 were those with recurrence ofsymptomatic pneumonitis. In all these cases, however, the first symptomshad occurred within 6 months after therapy.

Pro-inflammatory Cytokinies Markers: IL-1α and IL-6

We analyzed pro-inflammatory cytokine IL-1α, and IL-6 levels beforeradiation treatment, weekly during treatment, and up to 12 weeksfollowing radiation. FIG. 2 shows the absolute cytokine level (in pg/ml)(A1 for IL-1α, and A2 for IL-6) and the relative cytokine changesnormalized to individual pre-treatment value (B1 for IL-1α, and B2 forIL-6), as well as the comparison of absolute values between the groupswith and without radiation pneumonitis (C1 for IL-1α, and C2 for IL-6).The data showed a very wide range of individual circulatory IL-1α levels(A1), but a relative lack of changes with radiation treatment (B1). Incontrast to IL-1α, IL-6 levels were not as variable among individuals(A2), but they fluctuated somewhat with radiation. Of note, aftercompletion of radiation treatments, there is a trend toward an increaseof IL-6 in both absolute levels and relative changes (A2 and B2,p=0.065). Both IL-1α and IL-6 absolute levels were significantly higherbefore radiation, at multiple time points during radiation, and afterradiation (C1, and C2, p<0.05) in patients who subsequently developedradiation pneumonitis.

Pro-fibrotic Cytokine Markers: bFGF and TGF-β1

FIG. 3 demonstrates results of circulatory cytokine changes of fibroticcytokines bFGF and TGF-β1. Basic FGF levels fluctuated during treatmentsand showed no correlation with pneumonitis (A1, B1, and C1). Incontrast, there were many individual variations of circulatory TGF-β1(A1), but there was much lesser degree of relative changes duringradiation and after radiation up to 12 weeks post-therapy. Similar tobFGF, circulating TGF-β1 did not show an appreciable difference betweenthe group with and the group without pneumonitis (C2).

Chemokine and Adhesion Molecule Markers: MCP-1, L-Selectin, andE-Selectin

Plasma levels of MCP-1 (Monocyte Chemotactic Protein 1), L-Selectin, andS-Selectin (FIG. 4) were also measured. FIG. 4A demonstrates theabsolute levels of MCP-1 (A1), relative changes of MCP-1 (B1), and thecomparison of the groups with and without pneumonitis (C1). Our datashowed a decline of MCP-1 levels during the last week of radiation andup to 8 weeks after radiation (p<0.04). Data on L-Selectin demonstrateda marked and significant decline of the circulatory levels of L-Selectin(A2, p<0.01) and the relative changes (B2, p<0.01), and a lack ofdifference between the pneumonitis group and the non-pneumonitis group.There was some decline of circulatory MCP 1 near the end of radiation upto 8 weeks after treatments. Data on E-Selectin is similar to L-Selectinin that there was some decline of levels near the end of radiation andafter radiation (p<0.03) as well as a decrease of relative changesthrough most time points of the period investigated (p<0.01). There alsowas not a significant difference between the pneumonitis group and thenon-pneumonitis group.

Radiation pneumonitis and fibrosis can be regarded as the consequencesof a wound-healing inflammatory reaction to radiation damage of lungtissues. Research in immunological regulation of inflammation hasrevealed the complex interaction between local tissues and immune cellsmediated through chemokines, adhesion molecules, inflammatory cytokine,and fibrotic cytokines.

Inflammatory Cytokines and Radiation Pneumonitis

We have shown that lung radiation is associated with a temporalexpression of IL-1α, TGF-β1, collagen I, collagen III, and collagen IVgene expression in fibrosis-prone mice (C57BL/6). Among the panel ofcytokines potentially involved in the inflammatory response to radiationlung injury, IL-1α and IL-6 were the only two cytokines that correlatedsignificantly with radiation pneumonitis (FIG. 4). In addition,pre-treatment levels of both IL-1α and IL-6 were significantly higher inpatients who subsequently developed pneumonitis, supporting their roleas predictors of radiation pneumonitis. Our data showed some differencesbetween IL-1α and IL-6, however, in that when normalized to individualpre-treatment levels, IL-1α remains relatively constant during treatmentcourse, but there is a trend toward elevation of IL-6 at 8 to 12 weekspost-radiation.

The rise of IL-6 after completion of radiation was observed. Itcoincided with the period of clinical symptomatic pneumonitis and thisdeserves further investigation (FIG. 1). Both IL-6 and IL-1α areimportant immunoregulatory moieties. Although both are inflammatorycytokines, they differ somewhat in origin of cells and in somefunctional aspects. Both cytokines mediate fever and regulateinflammation and fibrotic response through immune cells. The source ofIL-1 is primarily from monocytes as well as alveolar macrophages. IL-6is synthesized by a variety of cells in the lung parenchyma, includingthe alveolar macrophages, type IT pneumocytes, T lymphocytes, and lungfibroblasts. In the in vitro system, when alveolar macrophages wereexposed to clinically relevant dose of radiation (2 Gy), it was foundthat both IL-1α and IL-1β were released in increased amounts. It hasbeen shown that IL-1 stimulates human lung fibroblast in the productionof IL-6 and stabilizes IL-6 messenger RNA production. In patients withhigher pre-treatment levels of IL-1α, IL-1α may regulate a subsequentincrease of IL-6 after radiation, was observed (FIG. 2).

Pro-fibrotic Cytokines and Radiation Pneumonitis

Pro-fibrotic cytokines participate in radiation lung injury, especiallyduring the development of lung fibrosis phase, which generally starts at4 to 6 months after treatment and continues without a clear end point.Lung fibrosis is equivalent to the scar after the initial inflammatoryphase of lung reaction to radiation injury. Although radiographicfibrosis in general is not observed until 4 to 6 months after completionof radiation, it has been reported that circulatory TGF-β1 changes mayserve as an early predictor for radiation pneumonitis and its expressionincreases with radiation in animal research models. Two pro-fibroticcytokines, bFGF and TGF-β1, and their changes in the association toradiation pneumonitis (FIG. 3) was investigated. As the incidence ofradiation pneumonitis peaks at 6 to 13 weeks in our cohort of patients,we analyzed our data up to 12 weeks and did not find an association inpredicting radiation pneumonitis with either bFGF or TGF-β1. Thisfinding may be attributed to the patient population and sample sizedifferences. Since cytokines are relatively fragile molecules, technicaldifferences in specimen collection, processing, and laboratory assaysmay also result in the differences in laboratory measurements.

We have discussed that circulatory measure of IL-1α and IL-6 turned aresignificantly associated with radiation pneumonitis. Thus, patients withhigher baseline levels of inflammatory cytokines are more vulnerable toradiation lung injury.

Figure Legends:

FIG. 1. Twenty-four patients were followed prospectively for clinicalsymptoms of radiation pneumonitis and radiographic changes. Thescattered plot demonstrates the time of either symptomatic pneumonitis(top line) or only radiographic infiltrates without symptoms (bottomline). Data showed that symptomatic pneumonitis was diagnosed primarilybetween 6 weeks and 13 weeks after completion of radiation with rareoutliers occurring prior to 6 weeks and between 6 months to 9 months.

FIG. 2. IL-1α absolute levels (A1), relative changes normalized toindividual pretreatment levels (B1), and the comparison of levelsbetween patients with grade 1 to 3 pneumonitis (solid bar) and nopneumonitis (hatched bar) are presented for pre-treatment baselinelevel, weekly during radiation, and up to 12 weeks after radiation. FIG.2 A2, B2, and C2 demonstrate the IL-6 absolute levels, and relativechanges and the comparison between the two groups of patients,respectively.

FIG. 3. Basic FGF a absolute levels (A1), relative changes normalized toindividual pretreatment levels (B1), and the comparison of levelsbetween patients with grade 1 to 3 pneumonitis (solid bar) and nopneumonitis (hatched bar) are presented for pre-treatment baselinelevel, weekly during radiation and up to 12 weeks after radiation. FIG.3 A2, B2, and C2 demonstrate the TGF-β1 absolute levels, relativechanges, and the comparison between the two groups of patients,respectively.

FIG. 4. Basic MCP1 absolute levels (A1), relative changes normalized toindividual pretreatment levels (B1), and the comparison of levelsbetween patients with grade 1 to 3 pneumonitis (solid bar) and nopneumonitis (hatched bar) are presented for pre-treatment baselinelevel, weekly during radiation and up to 12 weeks after radiation. FIG.4 A2, B2, and C2 demonstrate the L-Selectin absolute levels, relativechanges, and the comparison between the two groups of patients,respectively. In FIG. 4, A3, B3, and C3 demonstrate the E-Selectinabsolute levels, relative changes, and the comparison between the twogroups of patients, respectively.

EXAMPLE 2

Materials and Methods

Mice Strains and Radiation Treatment

Six to 7 week-old female C3H/HeN, BALB/c and C57BL/6 mice were used(Jackson Laboratories, Bar Harbor, Me.). The right hind leg (10 mice pergroup) was given 10, 20, 30, 40, 60, or 80 Gy in a single radiation dosewith a Shephered Irradiator, a 6000 Ci Cs source, together withcollimating equipment. The left, non-irradiated hind leg was used as thenon-irradiated control. Mice were sacrificed at different time pointsafter radiation (0.5, 1, 2, 4, 8, 12hrs, day 1, day 7, and day 14). Atleast 10 mice were used at each time point. Tissues from 3 mice wereused for histology, and the remaining animals were used for mRNAanalysis. Skin and muscle tissues from control and irradiated legs weredissected, and total RNA was isolated. Guidelines for the humanetreatment of animals were followed as approved by the University ofRochester Committee on Animal Resources.

Tumor Tissue RNA Isolation and RNase Protection Assays

Skin and muscle tissues from each treatment group (7-10 mice) werepooled and total RNA was isolated by pulverizing the frozen tissue anddissolving it in TRI Reagent (Molecular Research Center, Ohio) accordingto the manufacturer's specifications. To determine the integrity ofisolated RNA, 2 μg of RNA from each sample was fractionated on aformaldehyde gel and visualized by staining in ethidium bromide. RNaseprotection was performed using established multi-probes template sets(PharMingen, SanDiago, Calif.) as described previously. The interleukin(IL) sets include: IL-1α:, IL-1β, IL-1Rα, IL-6, IL-10 and IL-12. Twointernal controls, L32 and GAPDH, were used as loading controls. Thecocktail constructs were used to prepare P-UTP labeled antisense cRNAprobes using the PharMingen in vitro transcription kits (PharMingen,SanDiago, Calif.). Probes were hybridized with 30 μg of total RNA at 50°C. for 16 hr. RNase A (1 mg/ml) and RNase T1 (2000 U/ml) were then addedto digest single-stranded RNA. After digestion, the RNA was precipitatedand resuspended in gel loading buffer, heated at 95° C. for 5 min, andrun in 7% denaturing polyacrylamide gel (National Diagnostics, Ga.). Thegel was run for 2-3 hr at 60v, dried on Whatman filter paper, and placedon a phosphorimager screen for quantitative analysis using a CyclonePhosphorimager device (HP Company, Conn.). Area integration of eachmRNA-protected fragment was normalized against the protected internalcontrol band (GAPDH) in the corresponding lane to calculate the ratio oftargeted/GAPDH mRNA. In order to compare the basal levels withradiation-induced levels for each interleukine mRNA tested, relativemRNA levels (folds) were plotted. Some gels are shown with over-exposureof the control lanes to highlight differences in IL-1α/β expression.

Blood Cytokines Assays (ELISA)

Blood samples were collected from 3 mice strains at various time pointsafter radiation. After centrifugation for 30 minutes at 4° C., plasmaswere aliquated and stored at −70° C. until analysis. Immunoenzymetricassays for murine IL-1β (Endogen Inc, Cambridge, Mass.) were performedaccording to the manufacturer's instructions. A standard curve withcytokine-positive control was run in each assay and the lower limit ofdetection was determined to be 3.5 pg/ml. Most of non-irradiated micehad circulating IL-1β protein levels near the limit of detection.

In Situ Hybridization

Localization of the IL-1β gene in soft tissue was determined by in situlocalization and was performed as previously published. Briefly, legtissues were fixed in 10% formalin and 2% paraformadhyde by cutting thewhole leg into 3-5 pieces. Tissue sections were then placed on speciallyprepared slides (acid washed and T3-aminopropyl trietlioxysilane coated)and were deparaffinized and rebydrated. Proteinase K-digested sectionswere hybridized with appropriate amounts of IL-1β riboprobe. Sections tobe examined were hybridized with anti-sense RNA under conditions ofprobe excess, and, after washing, they were prepared for autoradiographyusing NBTII emulsion (Kodak, Rochester, N.Y.). After autoradiography andstaining, the slides were analyzed by bright and dark field microscopy.Backgrounds for these studies were determined using the sense stand RNAprobe. As positive controls for hybridization, some sections werehybridized with constitutively expressed mRNA (GAPDH) and were analyzedfor cell specific expression of the molecule of interest. Cell types andlocations of IL-1β over-expression were identified histologically.

Statistical Analysis

Cytokine mRNA expression levels from skin and muscle in non-irradiatedversus irradiated tissues were compared using the unpaired Student'st-test, or Mann-Whitney Rank Sum test as appropriate. Differences wereconsidered significant for p<0.05.

Results

Pathological Observation

At early time points after irradiation of the skin, the gross appearancewas only mildly different from one strain of mice to another. FIG. 5shows typical changes seen after 30 Gy. During this acute process, whichoccurs over the 14 days following limb irradiation, the C3H/HeN mice(least fibrosis sensitive strain) had some hair loss and leg swelling(FIG. 5 b). The BALB/c mice, which have intermediate fibrosissensitivity, had the most edema and hair loss (FIG. 5 f), while thefibrosis sensitive C57BL/6 mice had only a thinning of the fur withminimal edema over the first 14 days (FIG. 5 d). Local hair loss wasnoted during the first 14 days in all 3 mice strains, in a dosedependent manner. Ulceration was seen only in the high dose groups (60and 80 Gy) at 14 days, and it was less common in the C57BL/6 mice.However, the acute inflammation that occurred in these animals over the2 week observation period did not correspond to the degree of fibrosisthat is expected 2 months after therapy.

The histological changes mirrored the clinical examinations, with somequalitative differences. As an example, 30 Gy irradiated tissues atvarious times after treatment are shown in FIG. 6. C3H/HeN and BALB/cmice had lower basal densities of hair follicles compared to C57BL/6mice (FIG. 6 a, d, and g). Three days after 30 Gy irradiation, all 3strains had similar follicle densities; however, the sub-epidermalmatrix accumulation was more pronounced in BALB/c and C3H/HeN mice (FIG.6 c and f). At 14 days, inflammatory cells and fibroblasts in the dermiswere more pronounced in C57BL/6 mice (FIG. 6).

Expression of IL-1β mRNA

In order to determine the molecular correlation of radiation-inducedsoft tissue damage, we examined mRNA expression of interleukins, IL-1α,IL-1β, and IL-1Ra by RNase protection assay in skin and muscle tissuesfrom the 3 mice strains before and after different doses of radiation.As shown in FIG. 7 and FIG. 8, and summarized in Table 2, all 3 micestrains had detectable basal levels of IL-β mRNA in their skin (C3H/HeNmice had the highest), but only very low levels of IL-1β mRNA in muscle.Skin IL-1β mRNA expression was substantially increased within a fewhours following 30 Gy leg irradiation (FIG. 3). There were two phases ofIL-1β mRNA elevations: first from 0.5 to 4 hrs and then from 7-14 daysfollowing irradiation (FIG. 7 a, b, and c). C3H/HeN and BALB/c mice hadsimilar patterns of IL-β mRNA expression after irradiation (FIG. 7). Incontrast, the fibrosis-sensitive C57BL/6 mice had little if any IL-1βmRNA induction. Neither of the bimodal peaks were seen in irradiatedmuscle of C57BL/6 mice. Elevation of skin and muscle IL-1β mRNA inC3H/HeN and BALB/c mice was radiation dose-dependent. Very high dose (80Gy) radiation significantly increased mRNA expression of skin and muscleIL-β (6 and 9 fold, respectively) in C3H/HeN mice at 4 hrs (FIG. 8 a, b,and c). After 14 days, the 80 Gy dose significantly increased IL-1β mRNAlevels in both C57BL/6 and C3H/HeN mice, which were over 15-fold higherthan those of non-irradiated controls. Local leg radiation not onlyincreased local tissue IL-1β mRNA expression, but it also increasedcirculating IL-1β measured by ELISA. Circulating IL-1β was associatedwith tissue mRNA expression with bimodal elevations at 4-8 hr and againat 7 to 14 days in both BALB/c and C3H/HeN mice (FIG. 9 b). Thereappeared to be a dose response, with higher local radiation dosesleading to chronically higher circulating IL-1β levels. In order todefine the cell types producing IL-1β mRNA, in situ hybridization wasperformed on irradiated soft tissues. Increased IL-1β mRNA was mainlylocalized in keratinocytes and stroma cells in the dermis ofnon-irradiated skin.

Expression of IL-1α mRNA

As shown in FIG. 7 and Table 2, undetectable or very low levels of IL-1αmRNA were measured in the skin of C3H/HeN mice. A 2 to 6 fold inductionof skin IL-1α mRNA was detected in both C57BL/6 (FIG. 7 a) and BALB/cmice after high doses of radiation (FIG. 10 a and 10 b). This increasedskin IL-1α mRNA expression was radiation dose-dependent, progressed withtime, and was minimal at the sub-fibrogenic radiation doses (≦30Gy).Radiation did not appear to induce IL-1α mRNA expression in muscle ofany of the 3 mice strains (FIG. 10 c).

Expression of IL-1Ra mRNA

Like IL-1β mRNA, IL-1Ra mRNA was highly expressed in skin tissue, and nosubstantial difference in the basal levels of IL-1Ra mRNA was seen amongthe three strains (FIG. 11). Skin IL-1Ra, however, was dramaticallyinduced by radiation in C57BL/6 mice, but not in C3H/HeN or BALB/c mice.Induction of IL-1Ra mRNA in C57BU6 mice was radiation dose dependent.The effects of radiation on IL-1Ra mRNA expression in muscle of anystrain was minimal (FIG. 11 d).

Discusssion

Murine models were used to simulate the situation that occurs in humanskin after irradiation. This enabled us to examine the molecularcharacteristics of soft tissue fibrosis. Doses that caused little or nofibrosis (<30Gy), as well as highly fibrogenic doses (60-80Gy) were usedin the 3 mice strains. We expected that, if radiation-induced cytokinemRNA expression is a causal event, then high doses would induce higherlevels of cytokine mRNA, explaining strain variation in fibrosissensitivity. Two key questions were asked in this study: 1) Is there adifference in basal mRNA expression of certain cytokines in skin ormuscle tissues among 3 mouse strains with different fibrosissensitivities? 2) Does this difference in mRNA expression contribute tothe various radiation-related fibrosis responses in the three mousestrains? We demonstrated that: 1) skin tissues express higher levels ofseveral interleukins than muscle tissues, independent of mouse strain.This is consistent with prominent initial fibrosis occurring in thesubepidermal regions, with less and later development of fibrosis inmuscle tissue. 2) C3H/HeN mice have the lowest predisposition fordeveloping fibrosis and did not express IL-1α mRNA in their skin. Themost fibrosis sensitive strain, C57BL/6, had high basal andradiation-induced levels of this cytokine. Muscle, which is morefibrosis resistant than skin, also had lower or undetectable IL-1αexpression compared to skin. 3) Radiation induced elevation of IL-1βmRNA was biphasic with an early peak (1 to 4 hr) and another at a latertime (3 to 14 day). The first phase was absent in the fibrosis sensitivestrain, and it was intermediate in the strain with intermediate fibrosissensitivity. 4) Cytokine responses in muscle were more blunted, comparedto those in skin, and required higher radiation doses. 5) Cytokineresponses after local radiation could be large enough to be detected inthe circulation. 6) The cells synthesizing the greatest quantities ofIL-1β appear to be the keratinocytes and stromal cells of the epidermisand dermis. Taken together we propose that these patterns suggest thatbrisk IL-1α responses to radiation and high basal IL-1α mRNA levels areassociated with a higher risk for late radiation fibrosis. An earlypulse of IL-1β expression after irradiation appears to correlate with alower risk for developing radiation soft tissue fibrosis. The data alsoprovided evidence that circulating levels of cytokines might be a usefulmarker of local cytokine production following radiation.

It has been demonstrated both experimentally and clinically that highbasal levels of fibrogenic cytokines and/or growth factors are relatedto a higher incidence of radiation- or chemotherapy-induced late tissuedamage. Our recent animal studies also suggest that high blood TGF-βlevels are associated with a high risk for radiation-induced tissuefibrosis. We measured local and circulating levels of interleukin mRNAin our 3 mice strains with different fibrosis sensitivities becausehigher basal mRNA levels of these cytokines may also be related to ahigher risk of radiation-mediated normal tissue fibrosis. It is apparentfrom our data that C3H/HeN skin does not have detectable IL-1α mRNA. Lowor undetectable skin IL-1α mRNA in C3H/HeN mice, a fibrosis resistancemouse strain, may be responsible for its resistant phenotype. In ourradiation-induced lung fibrosis models, similar results were alsoobserved. The correlation of low mRNA levels of skin and lung IL-1α withincreased resistance of radiation-induced fibrosis warrants furtherinvestigation.

Radiation-induced expression of interleukin mRNA is organ-dependent. Allinterleukin responses were more pronounced in the skin than in muscle.Inducible levels of each cytokine, however, varied between skin andmuscle tissues. For example, radiation induced an elevation of skinIL-1α mRNA, not muscle IL-1α mRNA, in C57BL/6 mice. Our previous data incultured cell lines (keratinocytes, skin fibroblast, and squamous cellcarcinoma cells) also demonstrated that different cell types not onlyexpress different levels of each cytokine, but also respond to radiationdifferently. Our data here may also provide some guidance for clinicalradiation therapy. For example, avoidance of cutaneous radiation mightprevent cytokine cascades that could result in late tissue fibrosis.This is because soft tissue fibrosis begins in the subepidermis, laterextends through the dermis, and eventually involves the superficial andthe deeper muscle layers. Clinically, the efficacy of megavoltageradiation is in large part due to the lower epidermal dosimetry. It isan intriguing notion that patients with elevated basal IL-1α mRNA mightbe treated prophylactically with anti-cytokine therapy to preventfibrosis.

While radiation-induced alteration of interleukin mRNA in lung and otherorgans have been reported in several strains of mice, altered mRNAlevels of cytokines in soft tissues from different strains of mice havenot yet been reported. In this study, we collected and processed RNAsamples of three strains in the same RNase protection gel, and we alsocompared the IL-1 mRNA expression difference between skin and muscle. Wefound that the patterns of cytokine mRNA expression were consistent withthe degree of fibrotic response. In contrast, macroscopic andmicroscopic acute alterations were weak predictors of fibrosissensitivity. The lack of correlation between acute reactions and lateeffects has been studied for decades, and the role that cytokines andgrowth factors play appears to finally help explain the phenomenon.

Radiation increased IL-1 mRNA expression in two waves, the first atapproximately 4 hours after therapy and another 3 to 14 dayspost-radiation. Examination of corresponding skin tissue morphology ateach time point suggested that acute tissue response in preexistingcellular components may be responsible for the first peak of cytokineproduction. In situ hybridization studies suggest that keratinocytes,endothelial cells, and skin fibroblasts are the source of the earlyIL-1β mRNA expression. Infiltrating inflammatory cells and activatedfibroblasts are probably responsible for the second peak in cytokinemRNA production. Several studies have demonstrated that pulses of IL-1,given within 24 hours of radiation, are radioprotective. Endogenouspulsing of IL-1β in C3H/HeN mice after radiation may therefore partlyexplain this strain's higher resistance to fibrosis compared to C57BL/6mice.

In conclusion, we have shown that skin tissues produce more interleukinmRNA compared with muscle tissues. Skin IL-1α and IL-1Ra mRNA areupregulated in C57BL/6 mice, while IL-1β mRNA is increased in C3H/HeNand BALB/c mice within a few hours of local leg radiation. These resultsshow that radiation-induced differential mRNA expression for interleukinand varied basal levels of interleukin mRNA participate inradiation-induced normal tissue damage.

Legends

FIG. 5. Typical gross observation of radiation changes seen in control(a, c and e) and 14 days following 30 Gy irradiation (b, d and f) of theright hind limb in 3 mice strains. Edema was similar in all threestrains, and hair loss was similar in C3H/HeN and C57BL/6 mice, withslightly greater hair loss in BALB/c mice (f).

FIG. 6. The characteristic histological observation of progressivepathological changes of radiation fibrosis are shown in panels a throughi. Normal mouse skin for C3H/HeN (a), BALB/c (d), and C57BL/6 (g). Notethe thin epidermis with underlying papillary dermis, hair folliclescontaining multiple hairs. Leg muscle is free of significantinflammation. Day 3 (b, e, and h) and day 14 (c, f, and i) after 30 Gyradiation are shown. Early soft tissue reaction includes progressiveloss of dermal papilla, reduced hair follicle number, increased emptyhair follicles, and a superficial filling of the dermis with matrix andinflammatory cells. There is little inflammation of muscle, and thedermal inflammatory cell infiltrates were grossly similar in allstrains.

FIG. 7. IL-1β mRNA expression in irradiated limbs in 3 mice strains byRNase protection assay (a). IL-1β mRNA expression was quantitativelydetermined using a Cyclone PhosphorImager (HP Co, MI). IL-1β mRNA valuesare pooled from seven mice per measurement for irradiated skin (b) andmuscle (c). Lanes are shown over-exposed to demonstrate the absence ofIL-1α in the skin of C3H/HeN mice, and the brisk IL-1β response toradiation in C3H/HeN and BALB/c but not in C57BL/6. The early phase ofIL-1β mRNA expression was seen in muscle, while the later increase at 1to 2 weeks was less evident in muscle. 30 Gray is sufficient to cause ahigh frequency of severe acute reactions in all strains, but, at 2months following radiation, 30 Gy is sub-fibrogenic for most C3H/HeN andBALB/c mice.

FIG. 8. Determination of IL-1β mRNA expression in high dose (80 Gy)irradiated limbs from C3H/HeN and C57BL/6 mice by RNase protection assay(a and b). mRNA from seven mice was pooled. 80 Gy radiation inducedelevated IL-1β mRNA expression in both skin and muscle tissues. 80 Gy issufficient to cause substantial fibrosis and acute reaction in allstrains.

FIG. 9. Plasma IL-1β levels in C3H/HeN and BALB/c mice after limbirradiation. Circulating levels of IL-1β in platelet depleted plasmawere significantly increased after 30 Gy radiation in BALB/c mice(left). The difference from baseline was not significant at any timeafter 10 Gy, which is a sub-fibrogenic dose. In a separate experiment(right), 30 Gy radiation significantly increased blood IL-1β in bothC3H/HeN and BALB/c mice. The results suggest that circulating IL-1β is asurrogate for protein locally produced in the hind limb.

elevation compared to baseline significant p<0.05.

FIG. 10. Determination of IL-1α mRNA expression in 30, 40, or 60 Gyirradiated limbs from 3 mice strains by RNase protection assay. Eachvalue was normalized to its internal control GAPDH and represents thepooled expression from seven mice per measurement. Radiation elevatedIL-1α mRNA in skin (a and b) but not in muscle tissue (c). The effectwas greater with increased radiation dose. C3H/HeN mice express nodetectable IL-1α mRNA in their skin at any time after irradiation.

Elevation of IL-1α during the first day after radiation was mostpronounced in the fibrosis sensitive strain.

FIG. 11. Determination of IL-1Ra mRNA expression in 30, 40, or 60 Gyirradiated limbs from 3 mice strains by RNase protection assay. Eachvalue was normalized to its internal control L32 and represents thepooled expression from seven mice per measurement. Radiation-dose andtime dependant induction of IL-1Ra mRNA mainly occurred in skin, with nodetectable induction in muscle tissue. The fibrosis sensitive strain hadthe greatest induction of IL-1Ra.

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EXAMPLE 3

Material and Methods

Tumor Models and Radiation Treatment

Isotransplantable murine MCa-35 mammary tumor cells was inoculated i.m.into right hind thighs of 6-7 week-old female C3H/HeN mice (NCI,Fredrick, Md.). Right hind thigh tumors were given 60 Gy (single doseusing a Cs irradiator) when tumors reached 8-9 mm in diameter. Mice weresacrificed 20 days after radiation.

Tumors and the overlaying skin tissues were removed for histology andRNA preparation. Irradiated tissues (tumor and skin) were also collectedfor making paraffin blocks for immunohistochemical staining. Guidelinesfor the humane treatment of animals were followed as approved by theUniversity of Rochester Committee on Animal Resources.

Celebrex Treatment

Celebrex (Pfizer Inc.) powder was dissolved in PBS. Due to partialdissolution, the agent was mixed very well every time before gavaging.50 mg/kg (0.2 ml) Celebrex was given daily, and five days per week forconstitutive three weeks. Four experiment groups were used. All micewere treated with single 60 Gy radiation in tumor-bearing leg. Group 1was radiation alone; mice in group 2 were given 50 mg/kg Celebrex 2hours before radiation (2 hr pre-radiation); mice in group 3 and 4 werereceived the same amount of Celebrex at day 2 or day 7 post-radiation.Mice in the group 4 were received total 10 doses, and rest treated micewere given total 15 doses. All mice were sacrificed 20 day afterradiation.

Determination of Radiation Induced Skin Damage by 5-Scales ScoringSystem

Radiation induce skin damage was assessed using 5-scales Skin ScoringSystem. 20 days after single 60 Gy radiation, mice from each treatmentgroup were determined blindly for the degree of skin damage by threeinvestigators. Grade 1: normal skin; grade 2: slight hair loss inirradiated area; grade 3: radish and swollen tissue; grad 4: small areaerosion; grade 5: small ulceration. Grades 2-3 is referred as mild, andgrades 4-5 is considered as severe skin damage.

Tumor Tissue RNA Isolation and RNase Protection Assays

Total RNA was isolated from tumors and skin overlying tumors,respectively, with 9-10 mice in each treatment group by pulverizing thefrozen tissue, and dissolved in TRI Reagent (Molecular Research Center,Ohio) according to the manufacturer's specifications. To determine theintegrity of isolated RNA, 2 μg of RNA from each sample was fractionatedon a formaldehyde gel and visualized by staining in ethidium bromide.RNase protection was performed using established multi-probe templatesets (PharMingen, San Diego, Calif.) as described previously [Okunieff,1998 #4388]. The chemokine multiple templet includes: MCP-1, MIP-1α,MIP-1β, MIP-2, Rantes, Eotaxin and IP-10. The C-C chemokine receptormultiple templete includes: CCR1, CCR2, CCR3, CCR4 and CCR5. The C-X-Cchemokine receptor multiple templets includes: CXCR2 and CXCR4. Twointernal standards, L32 and GAPDH, were used as loading controls. Thecocktail constructs were used to prepare ³²P-UTP labeled antisense cDNAprobes using PharMingen in vitro transcription kits (PharMingen, SanDiego, Calif.). Probes were hybridized with 30 μg of total RNA at 50° C.for 16 hrs RNase A (1 mg/ml), and RNase T1 (2000 U/ml) was then added todigest single-stranded RNA. After digestion, the RNA was precipitatedand resuspended in gel loading buffer, heated at 95° C. for 5 min, andrun on a 6M urea, 7% denaturing polyacrylamide gel (NationalDiagnostics, Ga.). The gel was dried on filter paper and placed on aphosphorimager screen for quantitative analysis of mRNA expressionlevels for each cytokine/chemokine. Area integration of eachmRNA-protected fragment probe was normalized against the protected bandfor GAPDH or L32 mRNA in each corresponding lane to calculate the ratioof targeted mRNA/GAPDH mRNA expression. In order to compare the basallevels of each gene tested, relative levels (ratios) were plotted.

Quantitative Measurement of Total Structural and Perfused Vessels

Immunohistochemistry methods have previously been described in detail.Immediately following cryostat sectioning, tissue slices (normal muscleand tumor) were stained with CD31 antibody (PharMingen Calif.) fordetermination of total vasculature. The stained sections were imagedusing an epi-fluorescence equipped microscope, digitized (3-CCD camera),background-corrected, and image-analyzed using Image Pro software (MediaCybernetics, Mass.) and a 450 MHz Pentium computer. Color images fromindividual microscope fields were automatically acquired and digitallycombined to form four montages of the tumor cross-section (totalarea=15.5 mm2) using a motorized stage and controller. The imagemontages were processed to enhance the contrast between background andCD31 staining. From the enhanced images, locations of CD31-stainedvessels were recorded. The quantitative vascular information wasanalyzed using custom Fortran programs to perform a “closest individual”analysis as previously described. Briefly, the distances fromcomputer-superimposed sampling points to the nearest blood vessel weredetermined. The cumulative frequency distribution of these distancesprovided the probability of encountering vessels within any specifieddistance from the tumor cells. Median distances (μm) to the nearestvessel were used for statistical comparisons.

Statistical Analysis

mRNA levels (ratios) of tumors and skin from irradiated ornon-irradiated mice were evaluated using the unpaired Students t-test orMann-Whitney Rank Sum test as appropriate. Differences were consideredsignificant for p<0.05.

Results

20 days after single 60 Gy irradiated MCa-35 tumor skin had variedlesions including edema, erosion and superficial necrosis in most ofsaline-treated control mice 20 days after radiation (FIG. 12 a).However, Celebrex-treated tumors had less radiation-induced skin damagecompared with saline-treated controls (FIG. 12 b-d). The most ofCelebrex treated mice, regardless pre- (2 hr before radiation) orpost-radiation (day 2 or day 7 after radiation) had less inflammationand cellular component infiltration in the dermis (FIG. 13 b, c and d)compared with saline-treated controls (FIG. 13 a). 23.8% (5/21) of micein 60 Gy alone treated group developed severe skin damage, but only17.6% of mice in the pre-2hr Celebrex treated group, 5.3% of mice in thepost-day 2 Celebrex treated group, and 11.1% of mice in post-day 7Celebrex-treated group, appeared as the severe skin damage 20 days afterradiation. Oral administration of Celebrex also caused the reduction ofblood vessels in MCa-35 tumor (FIG. 12 f), focal necrosis (FIG. 12 g)and even massive tumor necrosis (FIG. 12 h) in some areas of tumors,compared with saline-treated controls (FIG. 12 e).

Because radiation inducing soft tissue damages has been reported toassociate with the persistent overproduction of cytokine or chemokine inirradiated normal or tumor cells, we next examined the effects ofCelebrex on the radiation-induced mRNA expression of chemokinesincluding five C-C family members (Rantes, eotactin, MIP-1α, MIP-β andMCP-1), one C-X-C family (MIP-2) and one C family member (lymphotactin),as well as C-C receptors (CCR1, CCR2 and CCR5) and C-X-C receptors(CXCR2 and CXCR4) in tumor skin and tumor tissues by RNase protectionassay. As shown in FIG. 14 and summarized in Table 3 and 4, Celebrextreatment caused the significant reduction of Rantes (2.3±1.1 vs7.4±1.6, P<0.05) and MCP-1 (10.2±1.1 vs 18.8±3.2, p<0.05) mRNAexpression in irradiated skin tissues, but not in tumor tissues (Table3), Although radiation induced higher levels of skin MIP-2 mRNAexpression in 37.5% (⅜) of mice, only 14.3%-28.6% of tumor skin had highMIP-2 mRNA expression after Celebrex treatment. Similarly,Celebrex-treatment did not significantly alter the tumor MIP-2 mRNA(Table 4). Celebrex not only reduced C-C chemokines, it also caused thedecrease mRNA expression of both C-C and C-X-C chemokine receptors intumor skin (FIG. 14B and D), not in tumor tissues (FIG. 14C). Allquantitative measurement are shown in Tables 3 and 4.

Due to each individual mouse variation, there was 15-30% ofCelebrex-treated mice still developed the moderate or severe skin damageafter radiation. Radiation-induced skin damage was quantitativelydetermined by the skin scores from each individual mouse. In order tofind out the relationship between overexpression of chemokines or theirreceptors mRNA and radiation-induced skin damages, the correlation ofskin scores and skin tissue chemokine and chemokine receptor mRNAexpression levels from each individual mouse were plotted and shown inFIG. 15. Significant positive correlation between skin damages (skinscores) and overexpression of chemokine and its receptor mRNA expressionwere observed in 60 Gy radiation-treated mice. However, the correlationof Celebrex-mediated the reduction of chemokine and chemokine receptormRNA expression with skin damages only occurred in Rantes (FIG. 15 a)and it receptor CCR5 (FIG. 15 d), MCP-1 (FIG. 15 b) and its receptorCCR2 (FIG. 15 d). Although Celebrex-mediated reduction of MIP-2 mRNAexpression did not correlate with less skin damage, related CXCR4 mRNAexpression was significantly reduced in Celebrex-treated mice, which hadless radiation-induced skin damage.

As shown in FIG. 16, Celebrex-treated mice had less infiltration ofinflammatory cells in the dermas (FIG. 16 c) compared with salinecontrols (FIG. 16 a). However, the infiltration of inflammatory cells intumor tissue was not obviously altered by Celebrex treatment (FIG. 16 band d).

Discussion

Thus we have discussed that: 1) Radiation induced Rantes/CCR5 andMCP-1/CCR2 mRNA expression was decreased by Celebrex; and 2)Celebrex-mediated reduction of chemokine and their receptor mRNAexpression was correlated with ameliorated skin damage. 2

1. A method for prophylactically treating radiation toxicity in normaltissue of a subject comprising administering an anti-radiation toxicityeffective amount of a cytokine blocking agent to said subject.
 2. Themethod of claim 1 wherein said cytokine blocking agent is administeredto said subject prior to said subject receiving radiation therapy. 3.The method of claim 1 wherein said cytokine blocking agent isadministered to said subject simultaneously with radiation therapy. 4.The method of claim 1 wherein said cytokine blocking agent comprisesanakinra or a MCP-1 blocker or a TGF-beta blocker.
 5. The method ofclaim 1 wherein said cytokine blocking agent is administered orally orparentally.
 6. The method of claim 6 wherein said parentaladministration is by intravenous infusion.
 7. The method of claim 1wherein said cytokine blocking agent blocks activity of cytokine IL-1.8. The method of claim 1 wherein said cytokine blocking agent blocksactivity of IL-1α or IL-1β.
 9. A method for prophylactically treatingradiation toxicity in normal tissue of a subject comprising regulatingcytokine IL-1 activity in said subject to decrease the radiationtoxicity.
 10. A method for prophylactically treating radiationpneumonitis, dermatitis, soft tissue fibrosis or central nervous systemtoxicity in a subject comprising administering an anti-radiationpneumonitis effective amount, an anti-radiation dermatitis effectiveamount, an anti-soft tissue fibrosis effective amount, or ananti-central nervous system toxicity effective amount of a cytokineblocking agent to said subject.
 11. A method for prophylacticallytreating radiation pneumonitis, dermatitis, soft tissue fibrosis orcentral nervous system toxicity in a subject comprising administering ananti-radiation pneumonitis effective amount, an anti-radiationdermatitis effective amount, an anti-soft tissue fibrosis effectiveamount, or an anti-central nervous system toxicity effective amount ofanakinra, a MCP-1 blocker, or a TGF-beta blocker.
 12. A method fortreating radiation toxicity in normal tissue of a subject comprisingadministering an anti-radiation toxicity effective amount of a cytokineblocking agent to said subject subsequent to subjecting said subject toradiation therapy.
 13. A method for diagnosing the likelihood for theoccurrence of radiation toxicity in the normal tissue of a subjectcomprising measuring the amount of cytokine IL-1, IL-160 , IL-1β or IL-6cytokines in the subject, the relative amount of such activity beingindicative of the likelihood of the subject experiencing an adversedegree of radiation toxicity when subjected to therapeutic radiation.14. A method for prophylactically treating radiation toxicity in thenormal tissue of a subject comprising first diagnosing the likelihoodfor the occurrence of radiation toxicity in the normal tissue of asubject comprising measuring the amount of cytokine IL-1, IL-1α, IL-1βor IL-6 cytokines in the subject, the relative amount of such activitybeing indicative of the likelihood of the subject experiencing anadverse degree of radiation toxicity when subjected to therapeuticradiation and then administering an anti-radiation toxicity effectiveamount of a cytokine blocking agent to said subject diagnosed as beinglikely to experience an adverse degree of radiation toxicity whensubjected to therapeutic radiation.